Malachite green is a chromophore with the unique ability to produce hydroxyl radicals upon intense laser irradiation. Chromophore-assisted laser inactivation (CALI) using malachite green (MG)-tagged antibodies has been applied to inactivate target proteins in a highly restricted manner, probing their temporally- and spatially-resolved functions. Application of this technique in a range of cell types and organisms has yielded detailed information about protein function that could not be obtained by conventional methods. In an attempt to implement this technique at the RNA level and achieve the same localized inactivation, a malachite green binding aptamer has been selected. 8 cycles of SELEX has given rise to approximately 30 molecules with varying affinities for the chromophore. Among these, one binder has been designated as the main aptamer based on its superior binding properties. Extensive site directed mutagenesis has shown that the aptamer consists of an asymmetric internal loop flanked by two A-type helices and that the binding activity is fully eliminated with any single nucleotide mutation in the internal bulge region. In vitro CALI experiments performed on the best binder have shown that the aptamer is cleaved specifically in the internal bulge at a uridine residue in a dose dependent fashion (co-incubated unspecific RNA molecules remain unaffected). The introduction of the MG aptamer into the 5' UTR of the S. cerevisiae Clb2 gene (a cyclin required for the cell cycle G2 to M transition) causes an elongated phenotype that becomes more pronounced upon addition of the ligand to the media. Analysis of mRNA and protein expression levels from cells in the presence and absence of malachite green show that the elongated phenotype is due to the reduced efficiency of translational initiation. NMR analysis of the major clone shows that the aptamer undergoes a structural reorganization in the presence of the cognate ligand as a result of adaptive binding. In vitro binding experiments with other compounds structurally related to malachite green have shown that the fluorophore tetramethyl rosamine (TMR) binds the MG aptamer with an affinity of 40 nM. Crystals of the MG aptamer complexed with TMR have been obtained and the crystal structure of the complex has been solved to 2.8 A resolution. Further site-directed mutagenesis made based on the crystal structure have shown that the aptamer utilizes an intricate network of noncanonical-base pairing and stacking interactions to create a very well defined pocket.
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